This proposal addresses the question of control of mammalian chromosome replication during early development. What DNA sequences are responsible for eukaryotic DNA replication, what role transcriptional regulation plays in controlling DNA replication, what similarities exist between initiation of cellular and viral DNA replication, and, does DNA replication during embryogenesis follows the same rules as DNA replication in differentiated cells? DNA or DNA-protein complexes will be injected into the nuclei of mouse 1 or 2-cell embryos, allowing embryonic development to continue in vitro while samples are analyzed to determine the role of specific DNA sequences and proteins in promoting DNA replication and gene expression. We have begun our studies with polyoma virus (PyV) and simian virus 40 DNA because they represent well characterized genomes that normally replicate in mouse and monkey cells, respectively, and, with the exception of viral T-antigen, rely exclusively on the host to carryout transcription and replication of their DNA. We have demonstrated that sequence-specific DNA replication can occur in preimplantation mouse embryos (in contrast to the sequence-independent replication observed in amphibian and sea urchin embryos), and that regulation of transcription in mouse oocytes and embryos can different significantly from that observed in embryonic and differentiated cell lines. We will extend these observations in four ways. (i) Determine the DNA sequence requirements for PyV DNA replication and gene expression in mouse preimplantation embryos, and relate them to the requirements in differentiated cells. In what way do gene enhancers and 'silencers' determine cell specificity for origins of replication? (ii) Determine the requirements for efficient DNA replication in embryos. How do PyV enhancer mutations affect DNA replication? What limits the ability of embryos to replicate DNA? (iii) Isolate mammalian chromosomal origins of replication by searching for cellular sequences that allow plasmid or phage vectors to replicate in mouse embryos. (iv) Identify proteins in differentiated mouse cells that activate origins of replication or enhancer elements that are normally inactive in embryos injecting nucleoprotein complexes assembled in differentiated cells into embryos and then measuring their ability to replicate and/or express genes.